US7691971B2 - Protein NMB1125 and use thereof in pharmaceutical formulations - Google Patents

Protein NMB1125 and use thereof in pharmaceutical formulations Download PDF

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US7691971B2
US7691971B2 US10/580,508 US58050804A US7691971B2 US 7691971 B2 US7691971 B2 US 7691971B2 US 58050804 A US58050804 A US 58050804A US 7691971 B2 US7691971 B2 US 7691971B2
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protein
nmb1125
vaccine
outer membrane
proteins
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US20070218000A1 (en
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Rolando Pajon Feyt
Enrique Guillen Nieto Gerardo
Gretel Sardinas Garcia
Nunez Lazaro Hiram Betancourt
Serra Lila Rosa Castellanos
Negrin Yasser Perera
Diaz Darien Garcia
Perez Olivia Niebla
Menendez Evelin Caballero
Blanco Sonia Gonzalez
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Centro de Ingenieria Genetica y Biotecnologia CIGB
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention is related to field of medicine, particularly to the development of new vaccine formulations of preventive or therapeutic application, which allows an increase in the quality of the immune response against vaccine antigens of diseases from different sources.
  • Neisseria meningitidis a Gram-negative diplococcus which only known host is man, is the causal agent of meningococcal meningitis. Usually this bacterium is found in asymptomatic carriers among the normal population, being this niche the most common source for its microbiological isolation.
  • Untreated meningococcal disease has a fatal course for most affected individuals, and vaccination could prevent this situation, by halting the events as early as at the bacterial colonization phase.
  • capsular polysaccharides as vaccine candidates.
  • a tetravalent vaccine, based on capsular polysaccharides, conferring protection against serogroups A, C, Y, and W-135 has been licensed in United States.
  • Antibodies elicited after vaccination are serogroup-specific (Rosenstein N. et al. 2001. Meningococcal disease . N. Engl. J. Med, 344, 1378-1388).
  • Serogroup B which is different from the rest, continues to be a significant cause of endemic and epidemic meningococcal disease, and this is mainly due to the complete lack of efficient vaccines against it. It has been noted that capsular polysaccharide B is poorly immunogenic, plus the existence of the theoretical risk for a vaccine based on this compound to induce immuno-tolerance and autoimmunity because of its structural similarity to oligosaccharide chains that are present in human neural fetal structures. (Finne J. et al. 1987. An IgG monoclonal antibody to group B meningococci cross - reacts with developmentally regulated polysialic acid units of glycoproteins in neural and extraneural tissues . J. Immunol, 138: 4402-4407). Therefore, the development of vaccines against serogroups B is concentrated in the use of sub-capsular antigens.
  • the OMV vaccine produced by the Finlay Institute in Cuba (commercially marketed as VA-MENGOC-BC) is produced from strain B:4:P1.19,15 with serogroup C polysaccharide and a preparation of high molecular weight OMPs and is adsorbed to aluminium hydroxide (Sierra G V et al. 1991. Vaccine against group B Neisseria meningitidis: protection trial and mass vaccination results in Cuba . NIPH Ann Dis. 14(2):195-210). This vaccine contributed to the rapid decline of the epidemic in Cuba (Rodriguez A P, et al. The epidemiological impact of antimeningococcal B vaccination in Cuba. 1999. Mem Inst Oswaldo Cruz. 94(4):433-40).
  • the vaccine produced by the Norwegian National Institute for Public Health (NIPH) was similarly intended initially for use during a period of hyperendemic disease caused by another organism from the ET-5 clone (B:15:P1.7,16). It was also a monovalent vaccine produced from purified outer membrane vesicles adsorbed onto aluminium hydroxide (Bjune G, et al. 1991. Effect of outer membrane vesicle vaccine against group B meningococcal disease in Norway . Lancet. 338(8775):1093-6).
  • Outer membrane vesicle vaccines appear to effectively present outer membrane proteins in a sufficiently natural conformation to allow the generation of functional bactericidal antibodies, at least in teenagers and adults.
  • the antibody responses generated have also been shown to increase opsonophagocytosis of meningococci.
  • the precise formulation of the vaccines i.e. OMP content, LPS content and the presence or absence of adjuvant
  • Opsonin response in vaccinees as measured by chemiluminescence . APMIS. 99(8):769-72, Gomez J A, et al. 1998.
  • the antigenic profile of disease isolates also changes rapidly and a vaccine with coverage of only a limited number of selected strains is likely to become ineffective within a few years unless the vaccine composition is changed to mirror local epidemiology.
  • OMV vaccines have been used more widely than any other serogroup B vaccine and are potentially useful in the context of outbreaks of disease caused by a single PorA type.
  • PorA protein The prominence of PorA protein and the significant level of variability in this protein, which appears to undergo continuous variation both between and during outbreaks (Jelfs J, et al. 2000. Sequence Variation in the porA Gene of a Clone of Neisseria meningitidis during Epidemic Spread . Clin Diagn Lab Immunol. 7(3):390-5) in epitopes to which most of the bactericidal activity in post-vaccination (and post-disease) is directed enhanced concerns that protection offered by single strain (monovalent) OMV-based vaccines might be serosubtype restricted (i.e. dependent on The PorA type).
  • an OMV vaccine was developed in The Netherlands at RIVM that contained PorA proteins from six different prevalent pathogenic isolates (Van Der Ley P and Poolman J T. 1992. Construction of a multivalent meningococcal vaccine strain based on the class 1 outer membrane protein . Infect Immun. 60(8):3156-61, Claassen I, et al. 1996. Production, characterization and control of a Neisseria meningitidis hexavalent class 1 outer membrane protein containing vesicle vaccine . Vaccine. 14(10):1001-8). In this case the vaccine vesicles were extracted from two variants of the well-characterized H44/76 strain which had been genetically engineered lo express three separate PorA proteins.
  • outer membrane proteins can induce a functional immune response against serogroup B disease but that none of the vaccines so far developed are universally protective due to the great heterogeneity of the surface exposed regions of the outer membrane proteins.
  • OMV outer membrane vesicles
  • the modest cross-reactive immunity induced by the outer membrane vesicles (OMV) vaccines has fuelled the search for an outer membrane antigen (or group of antigens), which induces functional antibodies and which is present on all meningococcal strains.
  • Such antigens if they were present on all strains irrespective of serogroup, might form the basis of a truly universal meningococcal vaccine, which would eliminate the potential problem of capsular switching on pathogenic strains following polysaccharide vaccination.
  • TbpA class 5 proteins
  • NspA NspA
  • FetA iron regulated proteins
  • TbpB forms part of the transferrin binding complex with TbpA.
  • TbpA has both a greater functional role in iron binding (Pintor M, et al. 1998. Analysis of TbpA and TbpB functionality in defective mutants of Neisseria meningitidis . J Med Microbiol 47(9): 757-60) and is a more effective immunogen than TbpB.
  • NspA was detected by ELISA on 99.2% of tested strains from serogroups A-C using anti-NspA monoclonal antibodies (Martin D, et al. 1997. Highly conserveed Neisseria meningitidis Surface Protein Confers Protection against Experimental Infection . J Exp Med 185 (7): 1173-83). These monoclonal antibodies have been shown to be bactericidal against numerous strains of meningococci and are able to reduce meningococcal bacteraemia in a mouse model (Cadieux N, et al. 1999.
  • Single protein vaccines have been used in the field for decades and generally exhibit good stability. If presentation in the form of vesicles is required, to allow the antigens to remain membrane bound, stability and reproducibility may be difficult to guarantee.
  • the immunogenicity and reactogenicity of outer membrane vesicles may vary with alterations in the amount of protein and LPS removed in the purification processes. A substantial body of experience in vesicle production has accrued in OMV vaccine manufacture, however, and the currently produced vaccines are subject to thorough quality control. Construction of entirely synthetic liposome vesicles may allow further optimization and standardization of such vaccines (Christodoulides M, et al. 1998.
  • ORFs began identifying the open reading frames that were predicted to encode either membrane bound, surface exposed or exported proteins. They identified 570 such ORFs, amplified them via the polymerase chain reaction and cloned them into Escherichia coli to allow expression of the encoded proteins as either His-tagged or glutathione S-transferase fusion proteins (Pizza M, et al. 2000. Identification of Vaccine Candidates against Serogroup B Meningococcus by Whole - Genome Sequencing . Science 287 (5459): 1816-20). The 61% (350) of the selected ORFs were successfully expressed, those which failed to express were often those containing more than one hydrophobic trans-membrane domain (possibly excluding a number of outer membrane bound proteins).
  • the recombinant proteins were purified and used to vaccinate mice.
  • the immune sera were then assessed for surface binding to multiple meningococcal strains by enzyme linked immunosorbent (ELISA) assay and flow cytometry and for bactericidal activity against two strains using the serum bactericidal assay.
  • ELISA enzyme linked immunosorbent
  • Seven proteins were selected for further study on the basis of a positive response in all three assays.
  • Trial vaccine formulations using a number of these proteins in combination with adjuvants have been shown to induce significant bactericidal tires against the homologous meningococcal strain (MC58) in mice, but none of the proteins induced SBA litres as high as an MC58 outer membrane vesicle vaccine (Giuliani M M, et al.
  • Vaccine components may be selected more effectively once an understanding of the contribution of individual antigens to the pathogenesis of N. meningitidis has been gained.
  • the antigens themselves may make effective vaccine candidates or, alternatively, the attenuated mutants could be considered as vaccine constituents.
  • the use of vaccine candidates with a high degree of sequence conservation among several species of pathogenic microorganisms could provide a solution to the multiple diseases they might cause in the case that these candidates induce a convenient response through the action of the immune system.
  • the technical aim that this invention pursues is the development of vaccine formulations capable of increasing and/or broadening the induced immune response against different pathogens or against a wide range of individual pathogen variants being these pathogens of cancer, bacteria, viral or any other origin.
  • the NMB1125 protein as a component of a vaccine formulation with therapeutic or preventive character against the meningococcal disease or any infection caused by a member of the Neisseria genus.
  • the novel character of this invention consists in the use, previously unreported, of the NMB1125 protein in formulations with new properties, able to induce a systemic and mucosal immune response of broad-spectrum protection, due to the conserved character of this protein in different isolates of Neisseria meningitidis and Neisseria gonorrhoeae.
  • FIG. 1 Cloning vector pM100 employed in the cloning and expression of protein NMB1125.
  • pTrip tryptophan promoter
  • N-term P64k P-64k N-terminal fragment
  • T4 Terminator Transcriptional terminator T4 phage.
  • FIG. 2 Final construction of nucleotide sequence of the gene NMB1125 in pM100 vector.
  • FIG. 3 SDS-PAGE analysis of fractions obtained from cellular disruption. Lane 1, total cells; Lane 2, cellular pellet; Lane 3, supernatant.
  • FIG. 4 SDS-PAGE analysis of purification process of NMB1125 from the disruption supernatant. Lane 1, resultant protein; Lane 2, contaminant protein of lower molecular weight found in a different chromatography fraction. Lane 3, sample before application.
  • FIG. 5 Antibody levels (IgG) against recombinant protein NMB1125, obtained after mice immunization by intra-nasal or intra-peritoneal route. ELISA results are represented, and expressed, as the inverse of the highest dilution that duplicates the value of pre-immune sera.
  • FIG. 6 Western blotting of NMB1125 protein present in N. meningitidis OMVs using sera from immunized mice with the recombinant protein. The immuno-identified NMB1125 is highlighted.
  • FIG. 7 IgA antibody response against recombinant protein NMB1125, at mucosal level, in mice immunized by intra-nasal route. Results are presented as the inverse of the highest dilution that duplicates the value of pre-immune sera.
  • A IgA antibody response in saliva.
  • B IgA antibody response in lung washes.
  • FIG. 8 Results of homology searches between NMB1125 protein (query) and annotated sequences in genomes from different serogroups of Neisseria meningitidis (“Sbjct”) using BLAST.
  • FIG. 9 Recognition of NMB1125 protein in different strains of N. meningitidis , by sera elicited against the recombinant antigen. In the graphic only are shown the results obtained when using semi-purified protein by intra-peritoneal route, however a similar behavior was observed in the rest of the cases. Results are presented as the inverse of the highest dilution that duplicates the value of pre-immune sera.
  • FIG. 10 Comparison among the sera elicited by immunization with the protein obtained by two methods, administered by intra-peritoneal route, in the passive protection experiments against meningococcal infection, in the infant rat model.
  • FIG. 11 Recognition of NMB1125 protein and a panel of un-related antigens by generated mAbs (mAbs H8/92, 3H2764 and 7D2/15).
  • P1 Class 1 protein Neisseria meningitidis strain B:4:P1.15;
  • P64k E3 subunit of pyruvate dehydrogenase from Neisseria meningitidis ;
  • T.T tetanus toxoid;
  • HBsAg Hepatitis B surface Antigen.
  • FIG. 12 Recognition of NMB1125 protein by human convalescent sera from survivors of meningococcal disease. As negative control healthy donor sera were employed. Results are shown as the absorbance (492 nm) in an ELISA type assay.
  • FIG. 13 JY1 anti-peptide titers from the sera of animals immunized with either free peptide (JY1), recombinant protein (NMB1125) or the conjugate JY1-NMB1125.
  • prowl.rockefeller.edu/cgi-bin/ProFound The search was subscribed to the genes and derived protein sequences contained in the SwissProt database (ebi.ac.uk/swissprot/) and NCBI (http://www.ncbi.nlm.nih.gov/), considering the oxidation of methionines, deamidation and carboxyamidomethylation of cysteines as possible modifications to be encountered.
  • the NM1125 protein was selected to be evaluated as possible vaccine candidate, from which one peptide was identified by mass spectrometry.
  • a homology based search was carried out in the NCBI sequence data base using the BLAST program (Altschul S F, et al. 1990. Basic local alignment search tool . J Mol Biol 215:403-410, ncbi.nlm.nih.gov/BLAST/). The results obtained after this procedure were marked as homologous, and beside the proteins reported in the published Neisserial genomes, several gene products marked as hypothetical proteins from different organisms, like Ralstonia, Yersinia and Pseudomonas species, were recognized.
  • the pM-100 cloning vector was employed. This vector allows the cloning to be carried out using different restriction enzymes and the generation of high expression levels of heterologous proteins in the form of inclusion bodies in E. coli.
  • the pM-100 vector ( FIG. 1 ) have the following elements: tryptophan promoter, gene segment codifying for the 47 amino acid stabilizing sequence from Nt-fragment of P64 kDa from N. meningitidis strain B:4:P1.19,15, sequence of bacteriophage T4 transcriptional terminator, and the sequence of the gene that confers resistance to ampicillin as selection marker.
  • the SignalP World Wide Web server (cbs.dtu.dk/services/SignalP-2.0) was employed.
  • the PCR product was digested using BglII and XhoI restriction enzymes, and cloned into vector previously digested pM-100 cloning vector.
  • the final construction is showed in FIG. 2 , and the NMB1125 protein is expressed as a fusion protein to the Nt-segment of P64 kDa protein.
  • Sequencing of the cloned gene NMB1125 was carried out using ALFexpress II automatic sequencer (Thermo SequenaseTM CyTM 5 Dye Terminador Kit, Amersham Biosciences) and oligonucleotides 1573 (Seq. ID. No. 8) and 6795 (Seq. ID. No. 9), that bind the sequence of the P64 stabilizer and T4 transcriptional terminator, respectively.
  • the plasmid generated herein was designated pM-238 for its posterior use.
  • the GC366 E. coli strain was transformed by the chemical method with the pM-238 plasmid ( FIG. 2 ).
  • the expression experiment was carried out in minimal media (M9) (Miller J H. 1972. Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, NEW York, USA) supplemented with 1% glycerol, 1% casein hydrolisate, 0.1 mM CaCl 2 , 1mM MgSO 4 and 50 ug/mL ampicillin.
  • Bacterial cultures were incubated 12 hours at 37° C. and 250 rpm. Grown cultures were centrifugated and ultrasonic disruption of the cellular pellet was performed (IKA LABORTECHNIK).
  • the protein containing fraction was dialyzed against Buffer A (25 mM Tris-hydroxymethyl aminomethane) from which the NMB1125 protein was purified by ionic exchange chromatography using a monoQ 5/5 column (Amersham Biosciences) with a gradient from 0 to 100% of NaCl in 1 h [Buffer A as equilibrium buffer and Buffer B (Buffer A+1M NaCl) as gradient buffer] after which an 80% pure protein was obtained as it is shown in FIG. 4 .
  • Buffer A 25 mM Tris-hydroxymethyl aminomethane
  • Buffer B Buffer A+1M NaCl
  • mice Female Balb/C mice (8-10 weeks-old) were immunized, once divided in 4 groups of 8 mice, each. Three immunizations were applied by intra-nasal or intra-peritoneal route, with 15 days-interval in between. The protein administered by intra-peritoneal route was emulsified with Freund's adjuvant.
  • Table 1 is described the composition of the immunogens:
  • the antibody titers (IgG) against the recombinant protein and the homologous protein present in the bacterium were determined by an ELISA, in serum samples taken after the third inoculation.
  • FIG. 5 the antibody titers against the recombinant protein of individual animals are shown.
  • Antibody levels were determined after the second inoculation, although they were higher after the third inoculation.
  • the immunoidentification by Western blotting was done, where the respective protein band was recognized.
  • the groups immunized by intra-peritoneal route had titers significantly higher than those elicited by intra-nasal route.
  • FIG. 6 The sera obtained after the immunization with the recombinant protein recognized the natural protein present in a preparation of outer membrane protein (OMP) of strain CU385. These results are represented in FIG. 6 .
  • OMP outer membrane protein
  • FIG. 7 show only the groups immunized by intra-nasal route. An increase in the IgA titer was observed in the group that received the semi-purified protein.
  • Sequences in groups A and B have 100% identity with the sequence obtained for the gene codifying for protein NMB1125 (Seq. ID. No. 3), and 99% identity in serogroup C.
  • sequence of the referred gene was determined for 3 Cuban isolates (Seq. ID. No. 5-7), which belong to serogroup B (B:4:P1.19,15) and a sequence alignment was done by using the ClustaIX program (http://www.ebi.ac.uk/clustalw/). The results of the alignment show that there is a great conservation in the nucleotide sequence of the gene NMB1125 among the analyzed strains.
  • FIG. 9 shows the results obtained with the sera elicited against the semi-purified protein administered by intra-peritoneal route. As it is observed, the immune sera recognized the protein present in different strains, with levels similar to the one found in the strain CU385. The rest of the sera had a comparable behavior in this assay.
  • a protection assay was conducted in the infant rat model for meningococcal infection. Twenty four rats (5-6 days old) were divided in groups of 6 rats each.
  • the immunization was done in Balb/C (H-2 d , female, 5-6 weeks old) and 4 doses were applied as follows: On days 0, 15 and 30 of the immunization routine, 10 ⁇ g of antigen NMB1125 per mouse (total volume 100 ⁇ l), were administered by subcutaneous route, emulsified with Freund's Adjuvant; on day 50, 10 ⁇ g of antigen per mouse in Phosphate Buffered Saline (140 mM NaCl, 270 mM KCl, 1.5 mM KH 2 PO 4 , 6.5 mM Na 2 HPO 4 ⁇ 2H 2 O, pH 7.2) were administered by intra-peritoneal route. Blood extractions were done on days 0 and 45.
  • Splenocytes from the animal with the highest titer were fused with X63 Ag8 653 mouse myeloma cells.
  • the resulting hybridomas were isolated and screened according to standard procedures (Gavilondo J V. 1995. Anticuerpos Monoclonales: Teor ⁇ a y Práctica, Elfos Scientiae, La Habana, Cuba).
  • FIG. 11 shows the results obtained in this experiment, all together 3 positive clones were obtained (mAbs H8/92, 3H2/64 and 7D2/15) which specifically recognized protein NMB1125, and do not react neither with the amino acid sequence corresponding to the N-terminal of P64k, nor with the rest of the non-related antigens assayed.
  • bactericidal test was performed.
  • the bactericidal antibody titer was expressed as the reciprocal of the highest dilution of the antibodies tested that was able of killing 50% or more bacteria, two of the mAbs generated (3H2/64 and 7D2/15) had bactericidal titers higher than 1:128 against the homologous strain B:4:P1.19,15 and one mAb (H8/92) higher than 1:80. Moreover, they had titers higher than 1:64 against the heterologous strains B:15:P1.7,16 and C:2a:P1.5.
  • a SPOTScan assay was done. A set of overlapping peptides that span the sequence of the protein was synthesized on a cellulose membrane, which was incubated with pooled sera diluted 1:100. The antigen-antibody reaction was detected by the incubation with a conjugate anti-murine immunoglobulin G-alkaline phosphatase, followed by the addition of a solution that contained the substrate Bromo-chloro-indolyl-phosphate.
  • FIG. 12 shows the results obtained with 5 convalescent's sera in this assay. It can be seen that the human sera recognized the protein, which indicates that it is expressed during the meningococcal infection and it is immunogenic.
  • Protein NMB1125 as a Carrier for a Peptide
  • the recombinant protein NMB1125 was conjugated to a 15 mer synthetic peptide, derived from the V3 region of protein gp120 from HIV-1, isolate JY1. The conjugation was done by the glutaraldehyde method. Free JY1 peptide, the recombinant protein NMB1125 and the conjugate JY1-NMB1125, were administered to adult mice in a 3-dose schedule, where the immunogens were emulsified with Freund's Adjuvant. Two weeks after the third dose, serum samples were obtained from the immunized animals, and the samples were analyzed by ELISA to determine the anti-peptide antibody titers.

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WO1999057280A2 (en) * 1998-05-01 1999-11-11 Chiron Corporation Neisseria meningitidis antigens and compositions
WO2000066791A1 (en) 1999-04-30 2000-11-09 Chiron Corporation Neisseria genomic sequences and methods of their use

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PL1693378T3 (pl) 2010-03-31
EP1693378A2 (de) 2006-08-23
RU2336900C2 (ru) 2008-10-27
DE602004023419D1 (de) 2009-11-12
KR20060123759A (ko) 2006-12-04
US20070218000A1 (en) 2007-09-20
WO2005054281A3 (es) 2005-08-04
ES2334138T3 (es) 2010-03-05
AU2004294376A1 (en) 2005-06-16
EP1693378B9 (de) 2010-05-19
ATE444305T1 (de) 2009-10-15
AR047263A1 (es) 2006-01-11
NO20063020L (no) 2006-09-01
CU23237A1 (es) 2007-09-26
CA2547317A1 (en) 2005-06-16
CN1890260A (zh) 2007-01-03
NZ547520A (en) 2009-07-31
WO2005054281A2 (es) 2005-06-16
RU2006123434A (ru) 2008-01-10
ZA200604556B (en) 2007-03-28
EP1693378B1 (de) 2009-09-30

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